Abstract:

The invention features core-shell microsphere compositions, hollow
polymeric microspheres, and methods for making the microspheres. The
microspheres are characterized as having a polymeric shell with
consistent shell thickness.

Claims:

1. A hollow microsphere comprising a polymeric shell, wherein the
thickness of said shell varies less than 10%.

2. The microsphere of claim 1, wherein said shell thickness varies less
than 5%.

3. The microsphere of claim 1, wherein said shell thickness varies less
than 1%.

4. The microsphere of claim 1, wherein said shell thickness varies less
than 0.5%.

5. The microsphere of claim 1, wherein said shell thickness is in the
range of 100-1000 nm.

6. The microsphere of claim 1, wherein said shell thickness is in the
range of 150-250 nm.

7. The microsphere of claim 1, wherein said shell thickness is in the
range of 350-450 nm.

8. The microsphere of claim 1, wherein said shell thickness is in the
range of 550-650 m.

9. The microsphere of claim 1, wherein said microsphere is substantially
devoid of silica.

10. The microsphere of claim 1, wherein said microsphere comprises a pore,
said pore having a size in the range of 10 -500 nm.

11. The microsphere of claim 1, wherein said microsphere comprises an
organic dye.

19. The microsphere of claim 1, wherein said shell comprises a co-polymer
selected from the group consisting of styrene-PMMA, benzyl
methacrylate-PMMA, styrene-PHEMA, styrene-PEMA, styrene-methacrylate, and
styrene-butylacrylate.

21. The microsphere of claim l, wherein the microsphere is prepared by a
method comprising:providing a substrate comprising a plurality of
hydroxyl groups;attaching an initiator agent to said hydroxyl groups to
form attached initiator agents;reacting the attached initiator agents
with a polymerizable unit under living polymerization conditions to form
a polymer shell over said substrate, said polymerization being confined
to a surface of said substrate; andexposing said substrate to an etching
agent for a time sufficient to allow for removal of said substrate from
said polymeric shell to form a hollow microsphere.

22. The microsphere of claim 1, wherein the microsphere is prepared by a
method comprising:providing a microsphere substrate;contacting said
microsphere substrate with a polymer nanosphere to yield a colloidal
assembly;heating said assembly to yield a core-shell composite;
andexposing said composite to an etching agent for a time sufficient to
allow for removal of said core from said shell to form a hollow
microsphere.

Description:

[0001]This application is a divisional application of, and claims priority
to, U.S. patent application Ser. No. 10/823,367, filed Apr. 12, 2004,
which is a continuation of U.S. patent application Ser. No. 10/033,389,
filed Oct. 25, 2001, now U.S. Pat. No. 6,720,007, which claims priority
to U.S. provisional application 60/243,104, filed Oct. 25, 2000. The
entire contents of each of the aforementioned applications is
incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0003]The invention relates to microsphere particles.

[0004]Hollow microsphere particles have a wide variety industrial and
biomedical uses. However, the formation of uniform and regular shell
structures, as well as control over the shell thickness, is difficult to
achieve using present methods, thereby restricting the uses of such
particles.

SUMMARY OF THE INVENTION

[0005]The invention features hollow microspheres and core-shell
microsphere compositions with consistent shell thickness using methods,
which allow controlled formation of a polymeric shell. The thickness of
the polymeric shell preferably varies less than 10%, more preferably less
than 5%, more preferably, less than 1%, and most preferably less than
0.5%. The variability in the thickness of the polymeric shell is
determined by measuring the thickness at two or more points on the
microsphere and calculating % divergence.

[0006]Shell thickness is controlled by the length of polymerization and is
varied to provide microspheres for divergent applications such as drug
delivery or synthetic pigment preparation. Duration of the polymerization
step is directly proportionate to the length of the polymer chains, and
thus, shell thickness. Shell thickness is in the range of 100-1000 nm. In
preferred embodiments, the shell thickness is in the range of 150-250 nm.
Alternatively, the shell thickness is in the range of 350-450 nm or in
the range of 550-650 nm. Preferably, the microsphere is substantially
devoid of silica. For example, the microsphere contains less than 10%,
more preferably less than 5%, more preferably, less than 1% silica by
weight.

[0007]The microspheres contain pores. A pore is a void in the polymeric
shell through which a composition may gain access to the hollow portion
of the microsphere. The microspheres have a certain porosity, and the
porosity is varied depending on the size and composition of the substrate
used to make the sphere. Pore size is varied depending on the size and
nature of the composition to be loaded into the hollow center of the
sphere as well as by changing the amount of crosslinking agent added
during polymerization. For example, the addition of increasing amounts of
a crosslinking agent produces microspheres with decreasing pore size.
Pore size is also affected by the addition of a foaming agent, i.e.,
addition of a foaming agent during production of the shell increases pore
size. For example, a pore has a diameter in the range of 10-500 nm.

[0008]Microspheres are useful as synthetic pigments, drug delivery
vehicles, and protecting agents. For example, the microspheres are used
in place of titanium dioxide, i.e., as a synthetic pigment, because an
empty microsphere in solution appears white. Organic dyes are
encapsulated in a hollow microsphere to produce a synthetic pigment of a
desired color. Empty or dye-encapsulated microspheres have several
advantages over standard titanium dioxide-based paints or dyes, e.g.,
improved color clarity or trueness.

[0009]The microsphere is also useful as protecting agent. For example, a
light-sensitive compound (e.g., a photo-bleachable dye) is loaded into a
hollow microsphere to protect its degradation from exposure to light or
chemicals prior to use. The compound is released from protection by
disrupting the microsphere, e.g., by crushing the sphere or contacting
the sphere with a solvent.

[0010]In addition to industrial applications, microspheres are used as
delivery vehicles for therapeutic agents such as polypeptides,
antibodies, enzymes, small molecule drugs, or nucleic acids.

[0011]The nature of the polymeric shell is varied to accommodate various
uses of the hollow microspheres. The microsphere shell typically contains
styrene, methacrylate, or any polymer with a high glass-transition
temperature (Tg). The shell contains a polymer resulting from the
polymerization of one or more monomers selected from the group consisting
of acrylonitrile, styrene, benzyl methacrylate, phenyl methacrylate,
ethyl methacrylate, divinyl benzene, 2-hydroxyethyl methacrylate,
cyclohexyl methacrylate, p-methyl styrene, acrylamide, methacrylamide,
methacrylonitrile, hydroxypropyl methacrylate, methoxy styrene,
N-acrylylglycinamide, and N-methacrylylglycinamide. Alternatively, the
shell contains a copolymer (random or block) selected from the group
consisting of styrene-PMMA, benzyl methacrylate-PMMA, styrene-PHEMA,
styrene-PEMA, styrene-methacrylate, and styrene-butylacrylate. The
strength and durability of the polymeric shell is increased by
crosslinking polymer chains.

[0012]The invention also includes methods of making hollow microspheres by
providing a substrate containing a plurality of hydroxyl groups and
attaching an initiator agent to the hydroxyl groups to form attached
initiator agents. Any solid substrate, which is characterized as
containing hydroxyl groups on its surface and is dissolvable (following
polymerization of the shell) is suitable. For example, the substrate is
silica, alumina, mica, or a clay composition. Alternatively, the
substrate is a crystal, which has been coated with a silica. The
initiator agents react with a polymerizable unit under polymerization
conditions to form a polymer shell over the substrate. The polymerization
is confined to a surface of the substrate. A polymer chain is initiated
at the initiator agent and is extended away from the substrate during
polymerization. To remove the substrate from the polymeric shell (to
yield a hollow microsphere), the substrate is contacted with an etching
agent for a time sufficient to allow for elimination of the substrate
from the polymeric shell. An etching agent is a composition which removes
a solid substrate from the center of a polymer-coated substrate, leaving
a polymeric shell. Preferably, at least 85% of the substrate, more
preferably 95%, more preferably 99%, and most preferably 100% of the
substrate is removed from the core of the sphere. Etching agents include
bases or acids, e.g., hydrochloric acid (HCl), hydrogen fluoride (HF),
sulfuric acid (H2SO4), sodium hydroxide (NaOH), potassium
hydroxide (KOH). Alternatively, the substrate is metal, and the etching
agent is an oxidizing or reducing agent. For example, a silica substrate
or mica is removed by etching with HF, and an alumina or clay substrate
is removed by etching with KOH. Optionally, the method includes a step of
exposing the polymer shell to a crosslinking agent.

[0014]The invention also includes a core-shell composition. A core-shell
composition is a composition, which contains at least two structural
domains. For example, the core domain is encased in the shell domain, and
the shell domain is characterized as having different physical and
chemical properties than the core. The core portion contains a first
compound, and the shell contains a second compound (which is not present
in the core portion). The core and shell differ by the presence or
absence of at least one compound. A method for preparing a core-shell
composite includes the following steps: providing a microsphere
substrate; contacting the microsphere substrate with a polymer nanosphere
to yield a colloidal assembly; and heating the assembly to yield a
core-shell composite.

[0015]An alternative method for preparing a hollow microsphere includes
the following steps: providing a microsphere substrate; contacting the
microsphere substrate with a polymer nanosphere to yield a colloidal
assembly; heating the assembly to yield a core-shell composite; and
exposing the composite to an etching agent for a time sufficient to allow
for removal of a core composition, e.g., silica, from the shell polymer
composition to form a hollow microsphere.

[0016]A colloidal assemby is an organized structure of two or more
particle types. For example, the assembly is organized such that the
nanospheres are assembled onto the surface of a microsphere. Preferably,
the microsphere is 1-100 μm in diameter; more preferably, the
microsphere is less than 75 μm in diameter; more preferably, the
microsphere is less than 50 μm in diameter; and even more preferably
the microsphere is less than 25 tm in diameter. For example, the
microsphere is 3-10 μm in diameter. The nanosphere is 1-1000 nm in
diameter. Preferably, the nanosphere is less than 500 nm; more
preferably, the nanosphere is less than 250 nm. For example, the
nanosphere is 100-200 nm in diameter.

[0017]The nanospheres and/or microspheres are optionally modified to
contain a reactive substituent. Preferably, the microsphere and
nanosphere contain different substituents, which associate, bind, or
react with one another. For example, the nanosphere contains an
amine-modified polymer, e.g., an amine-modified polystyrene (PS), and the
microsphere comprises an aldehyde-modified composition, e.g.,
glutaraldehyde-activated silica. The microsphere substrate contains
silica, alumina, mica, or clay. In another example, the nanosphere
contains avidin and the microsphere contains biotin, or the nanosphere
contains biotin and the microsphere contains avidin. The nanosphere may
contain one type of polymer or a mixture of polymers. For example, the
nanosphere contains PS, PMMA, or both. The microspheres are optionally
contacted with a mixture of different nanospheres, e.g., a mixture of PS
nanospheres and PMMA nanospheres, to yield a composite polymer shell. The
ratio of different polymer nanospheres is varied to achieve a desired
effect, e.g., strength or porosity. For example, the ratio of PS: PMMA is
50:50, 100:1, 10:1, 5:1, or 2:1.

[0018]The colloidal assembly is heated to a temperature greater than the
T. of the polymer nanosphere to melt the polymer nanospheres. The polymer
flows over the microsphere surface resulting in an essentially uniform
coating, i.e., the thickness of the polymer shell varies less than 10%
over its entire surface. For example, the colloidal assembly is heated to
at least 100° C. To melt PS and/or PMMA nanospheres, the colloidal
assembly is heated to 170-180° C.

[0019]Other features and advantages of the invention will be apparent from
the following description of the preferred embodiments thereof, and from
the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIGS. 1A and 1B are scanning electron micrographs of hollow
microspheres. FIG. 1A is a micrograph of silanized silica microspheres,
and FIG. 1B is a micrograph of the same microspheres after coating with
poly(benzyl methacrylate) by controlled/living radical polymerization for
14 h.

[0021]FIGS. 2A and 2B are scanning electron micrographs of the polymer
microspheres after etching with HF. FIG. 2A is a scanning electron
micrograph of etched microspheres, and FIG. 2B shows the microspheres
dispersed in water to allow visualization of individual particles.

[0022]FIG. 3 is a scanning electron micrograph of the hollow polymeric
microspheres obtained by crushing the hollow spheres by applying physical
pressure after freezing in liquid nitrogen. Both broken and intact
polymer spheres are seen.

[0026]FIG. 7 is a linear graph of a FITR spectrum of the shell
cross-linked hollow poly(benzyl methacrylate) microspheres.

[0027]FIG. 8 is a graph of a tapping mode AFM scan of the surface of
hollow PBzMA microspheres.

[0028]FIG. 9 is a diagram showing a polymerization step for coating silica
particles.

[0029]FIG. 10 is a diagram showing a method for making hollow
microspheres.

[0030]FIG. 11 is a line graph showing release of fluoroscein from hollow
microspheres (dashed line) compared to release from coated beads over
time.

[0031]FIG. 12 is a diagram showing a scheme for assembling composite
materials via both glutaraldehyde chemistry and biospecific interactions.
The top section illustrates assembly starting with amine-labeled silica.
Glutaraldehyde treatment followed by reaction with amine-modified
polystyrene nanospheres results in a silica-polymer composite that can be
heated at 170-180° C. to melt the polystyrene. A core-shell
material composed of a silica core and polystyrene shell is produced. The
bottom section illustrates assembly of biotin-labeled polystyrene
nanoparticles onto avidin-coated silica microspheres.

[0033]FIG. 14 is a tapping mode scanning force microscopy (SFM) scan of
the surface of the composite produced when 200 nm PS nanospheres are
assembled on 5 μm diameter silica.

[0034]FIG. 15 is a scanning electron micrograph of a non-specific binding
control. 100 nm amine-modified nanospheres were mixed with amine-labeled
silica microspheres under conditions identical to those in the assembly
process.

[0037]FIG. 18 is a line graph showing a comparison of FTIR spectra of pure
polystyrene and PS-silica composite. The spectra shown are in the range
of 2850 cm-1 to 2950 cm-1. Peaks in this wavenumber range
correspond to the aliphatic C--H stretching of polystyrene.

[0038]FIG. 19 is a line graph showing FTIR spectra of different core-shell
composites. The top spectrum (PS/silica composite) has peaks
corresponding to both silica and PS, while the middle spectrum
(PS/PMMA/silica composite) has an additional peak corresponding to the
carbonyl of the PMMA polymer. The bottom spectrum is from plain silica
microspheres.

DETAILED DESCRIPTION

[0039]Hollow polymer microspheres were prepared by coating silica
microsphere templates with poly(benzyl methacrylate) using surface
initiated controlled/living radical polymerization and subsequently
removing the core by chemical etching. Shell thickness was controlled by
varying the polymerization time. Scanning electron microscopy was used to
characterize the products and demonstrate that the polymer microspheres
were hollow. FTIR spectroscopy showed that the silica cores were
completely removed by etching.

Surface Confined Living Radical Polymerization: A New Method for Preparing
Hollow Polymer Microspheres on Silica Templates.

[0040]Uniform hollow polymeric microspheres were made by using surface
confined living radical polymerization. Using the silica microsphere as a
sacrificial core, hollow microspheres were produced following core
dissolution. First, a controlled/living polymerization was conducted
using an initiator attached to the surface of silica microparticles to
initiate atom transfer radical polymerization (ATRP). This procedure
yielded core-shell microparticles with a silica core and an outer layer
of covalently attached, well-defined, uniform thickness polymers. The
silica cores were subsequently dissolved, resulting in hollow polymeric
microspheres. Silica microspheres were coated with polymer in two steps
(FIG. 9). For example, first a benzyl chloride monolayer was prepared by
silanization of silica microspheres. In the second step, surface-modified
silica microspheres were heated in presence of copper halide, complexing
agent (dipyridyl) and benzyl methacrylate monomer in xylene at high
temperature to prepare uniformly coated microspheres. Polymer coated
silica microspheres were then immersed in aqueous HF solution to yield
uniform hollow poly(benzyl methacrylate) (PBzMA) microspheres. In order
to confirm that the microspheres are hollow, the microspheres were first
frozen in liquid nitrogen and then crushed between two glass plates. FIG.
3 shows a SEM image of intact and broken hollow polymer microspheres.

[0041]The process by which hollow polymeric microspheres are made differs
significantly from methods known in the art. The method presented herein
for preparing uniform hollow polymeric beads utilizes surface confined
living radical polymerization technique on silica templates.

[0042]Earlier processes for making hollow polymer latex particles include
one developed by Rohm and Haas. This earlier process involved making
structured particles with a carboxylated core polymer and one or more
outer shells. The ionization of the carboxylated core with base expands
the core by osmotic swelling to produce hollow polymer particles. Another
method involved an emulsion polymerization of styrene containing a small
amount of vinyl carboxylic acid in the presence of a hydrocarbon,
surfactant, and a water miscible alcohol. These processes are complex and
involve several steps employing different chemistries. Other methods
involve the synthesis of hollow nanoscopic polypyrrole particles to
employ gold nanoparticles as a template from which to grow the polymer
shell, followed by dissolution of the template. The methods of the
invention offer several advantages over earlier know methods in that the
present process yields microspheres with relative uniformity of shell
thickness. Another advantage is that the shell thickness is reliably and
consistently controllable. In contrast, formation of uniform and regular
shell structures surrounding the particles, as well as control over the
shell thickness, has been difficult to achieve using the earlier methods,
because polymerization is not restricted to the surface of the templates.

[0043]The surface confined living radical polymerization method presented
herein is simple, flexible and enables control over the shell thickness
and composition by adjusting polymerization time and monomer
concentration. Unwanted solution phase polymerization is also prevented
using this method. This method is applicable for preparing variety of
hollow polymer microspheres. This approach may allow for the fabrication
of different shapes of hollow polymeric materials produced from a variety
of templates.

[0044]A wide range of monomers are used to make the shell of the hollow
microsphere. Examples of monomers, which are compatible with the living
polymerization procedure, are listed in Table 1.

[0046]As is described above, a silica particle serves as a scaffold upon
which the shell is built by polymerization. An initiator is attached to
the surface of silica microparticles to initiate atom transfer radical
polymerization (ATRP). Examples of Living Radical Initiators (which are
immobilized on microsphere surfaces by silanization or some other method
prior to living radical polymerization) include those listed in Table 3.

[0047]Hollow polymer microspheres are a class of materials which have
application in the fields of medicine and materials science. For example,
the microspheres are used for product encapsulation for controlled
release of drugs and dyes, protection of light sensitive compounds,
enzyme encapsulations, and adhesives.

[0048]Hollow microspheres are made in a wide range of sizes (inner/outer
diameter) to suit a particular application. Pore size may also be varied
to order. Pore size is controlled by varying the amount of crosslinker in
the polymerization mixture. Generally, the pore size of the hollow
microsphere is slightly larger than the pore size of the template due to
the etching process. A larger pore size is suitable for adhesive
applications whereas a smaller pore size suits encapsulation of enzymes
or other therapeutic agents. Spheres with a pore size of 100 nm to 500 nm
(e.g., a sphere, which has a diameter of at least 1 micron sphere in
which the diameter of the pore is greater than about 10% of the diameter
of the sphere) are useful in the formulation of adhesives.

[0049]Synthetic Pigments

[0050]Hollow polymer nanospheres and microspheres are useful in the
industrial production of paints and pigments. The microspheres are added
to convention paint formulations as extenders. Hollow polymer
microspheres and nanospheres (e.g., 300-500 nm outer diameter) are used
in the production of synthetic pigments. Hollow polymer particles with
outer diameters in the range of 1-5 microns in diameter have been
produced using the methods described herein. These particles are useful
in the production of synthetic pigments as well. Nanometer-scale hollow
spheres are produced by performing living polymerization with 300-500 nm
diameter silica templates, followed by silica etching in hydrofluoric
acid.

[0051]The method described herein for preparation of hollow polymer
microspheres may have several advantages over current methods for
preparing synthetic pigments. Since synthetic pigments are made from
hollow polymer spheres, the shell thickness and composition must be
controlled in some manner. Shell thickness is correlated with the opacity
of the resulting pigment. Template directed living polymerization allows
the polymer shell thickness to be more accurately controlled than in
other known methods. This results in better control of the opacity of the
resulting pigment. Polydispersity of the polymer in the spheres also
influences opacity. By using living polymerization methods,
polydispersity is also under better control and more consistent than in
other methods. Polydispersity also influences the uniformity of the
hollow microsphere surface.

[0052]Material Encapsulation/Drug Delivery

[0053]Hollow polymer microspheres also have application in the fields of
materials encapsulation and drug delivery. Drugs such as tranilast or
ibuprofen are encapsulated in polymeric microspheres. The spheres are
used to slowly release drug over time in the digestive tract.
Biocompatible hydrogels such as polyacrylamide-chitosan are useful for
sustained antibiotic release. For example, microspheres with a core size
of approximately 3 micrometers are used for drug delivery.

[0054]Microspheres produced by living polymerization are more advantageous
for drug delivery applications because of the consistency in shell
thickness and porosity. In contrast, the shell thickness of the
microspheres produced by existing technology cannot be controlled during
the polymerization. Being able to control the shell thickness and
therefore the rate of drug release from the microspheres is a significant
advantage of the microspheres of the invention.

[0055]Studies on encapsulation and release of test agents, e.g., a dye,
from hollow microspheres were carried out as follows. Crosslinked hollow
poly(benzyl methacrylate) or PBzMA coated silica (approximately 1 mg)
beads were soaked with fluorescein in a methanol solution overnight.
Excess fluorescein was removed by centrifugation (1000 rpm), followed by
a wash with methanol. The dye-loaded beads were immersed in 0.5 ml
methanol. Release of the dye from hollow microspheres was monitored by
measuring the increase in fluorescence of the surrounding solution as a
function of time. The data shown in FIG. 11 indicate that hollow
microspheres or beads (dashed line) are effectively loaded with a
composition of interest and that the composition is released from the
hollow microspheres into the surrounding environment in a time-dependent
manner. In contrast, the composition is not loaded (and therefore, not
released) from coated solid beads (solid line) under the same conditions.

[0056]Microspheres produced for delivery of therapeutic products are
washed with water or a physiologically-compatible buffer (e.g.,
phosphate-buffered saline) following the etching procedure to remove the
silica template and residual etching agent. The microspheres are then
contacted with a therapeutic agent in solution phase. The microspheres
are loaded with the agent by diffusion.

[0057]Block co-polymer hollow microspheres may be produced using the
living polymerization method. The composition of the blocks can be
tailored for particular drug delivery applications.

Protecting Agents

[0058]Hollow polymer microspheres are used as protecting agents to
stabilize materials from exposure to light, solvents or other exposures
to which they may be sensitive. For example, a sensitive composition is
loaded into the microspheres. The composition is protected from light or
exposure to other damaging agents until the microsphere is physically or
chemically disrupted and the compositions is released from the
microsphere.

[0059]Hollow microspheres are also used as coatings. The polymer used to
make the polymeric shell of the hollow microsphere is tailored to the
application desired. For example, acrylate or methacrylate polymers are
suitable for most coating applications. Such microspheres are useful as
sunscreen compositions. The microspheres are used alone or in combination
with standard sunscreen compositions.

[0060]Hollow microsphere coatings are applied to paper or photographs to
protect them from light-mediated aging.

Production of Hollow Microspheres

[0061]The process for making uniform hollow polymeric microspheres
utilizes surface confined living radical polymerization. Using the silica
microsphere as a sacrificial core, hollow microspheres are produced
following core dissolution. First, a controlled/living polymerization is
conducted using an initiator attached to the surface of silica
microparticles to initiate atom transfer radical polymerization (ATRP).
This procedure yields core-shell microparticles with a silica core and an
outer layer of covalently attached, well-defined uniform thickness
poly(benzyl methacrylate) (FIG. 9). The silica cores are subsequently
dissolved, resulting in hollow polymeric microspheres (FIG. 10).
Surface-initiated living polymerization is a polymerization process in
which control of molecular weight is controlled by adjusting the monomer
concentration and termination reactions are substantially eliminated.
Polydispersity is thus lowered, enabling fine control of shell thickness,
i.e., the shell surfaces are more uniform. Shell thickness and variations
in shell thickness between two or more locations of the sphere are
measured using methods known in the art.

[0062]In comparison to standard solution or bulk living polymerization,
surface-initiated living radical polymerization has several advantages.
The growing radicals generated on the surface are not easily terminated
by bimolecular reactions due to limitations of the solid surface on which
the polymer chains are chemically attached, the low free radical
concentration and the low mobility. By using a controlled living
polymerization procedure to covalently attach polymer chains to
microsphere surfaces, one can control the thickness and uniformity of the
coated polymer film. Unwanted solution phase polymerization is also
prevented using this method. An additional benefit is the ability to
prepare block copolymers by the sequential activation of the dormant
chain end in the presence of different monomers. Although grafting of
polymers on flat and porous silica surfaces by using living radical
polymerization (von Werne et al., 1999, J. Am. Chem. Soc. 121:7409-7410)
has been described, the procedure for making uniform hollow polymeric
beads by using the living radical polymerization technique on a silica
microsphere template is completely new. The process of von Werne et al.
is a method of making a composite film by grafting flat and porous silica
surfaces particles using living radical polymerization.

[0063]Rather than producing microspheres, the method of von Werne et al.
yields a hexagonally-ordered film with embedded silica nanoparticles. In
contrast, the inventive method yields hollow microspheres with shells of
consistent thickness.

[0067]The reactions for bead coating consisted of two steps, which are
schematically shown in FIG. 9. The silica particles were first cleaned
with acetone several times to remove potential impurities. A benzyl
chloride monolayer was prepared by silanization of silica microspheres. A
mixture of 0.9 ml of acetone and 0.1 ml of CTMS were added to 6-7 mg of
purified silica microspheres in a 1.5 ml polypropylene microcentrifuge
tube. The bead suspension was shaken at room temperature for 2 h in the
dark. After silanization, the silica beads were separated from the
suspension by centrifugation, washed with acetone to remove unreacted
silane coupling agent and then cured at room temperature overnight in the
dark.

[0068]In the second step, the living radical polymerization was performed.
A 4 ml glass vial was charged with 6-7 mg of the silanized silica
microspheres and 0.75 ml of dry p-xylene. Dry argon gas was bubbled
through the mixture for 15 min to remove oxygen from the polymerization
system. After the removal of oxygen, 0.0067 g (0.068 mmol) of CuCl,
0.0316 g (0.21 mmol) of 2,2'-dipyridyl and 0.75 ml of benzyl methacrylate
were added to the same reaction mixture. The vial was then sealed with a
high temperature silicone rubber septum and argon was bubbled through the
mixture for another 20 min. to ensure that oxygen was removed completely.
The mixture was sonicated for 1 min to accelerate dissolution into
xylene. The reaction was heated with constant stirring (with a magnetic
stir bar) at 105-110° C. using a silicone oil bath. Polymerization
time was varied from 1 to 14 h to produce polymer shells with different
thicknesses. After polymerization, the coated microspheres were separated
from the suspension by centrifugation, and then washed several times by
centrifuging/resuspending in THF and methanol. Cross-linked polymer
shells were prepared by adding 10% ethylene glycol dimethacrylate (with
respect to benzyl methacrylate monomer) into the above mixture. The rest
of the procedures were the same as those for linear polymerization.

[0069]Procedure for Making Hollow Polymeric Microspheres

[0070]The synthesis of hollow polymeric microspheres is schematically
represented in FIG. 10. Briefly, PBzMA coated silica particles were first
suspended in tetrahydrofuran (THF). The bead suspension was filtered
through a 0.5 micron pore size Fluopore membrane (Millipore Corporation,
Bedford, Mass.). A thin pellet of coated microspheres was formed on the
top of the membrane. The product was dried in an oven at 60° C.
for 2 h. A 10% aqueous hydrogen fluoride (HF) solution was prepared by
diluting 50% HF with ultra-pure water. The membrane containing the pellet
was placed in a small polystyrene Petri dish and then 3.25 ml of 10% HF
solution was added to immerse the pellet. The reaction was allowed to
continue for 3 h at room temperature to etch the silica cores completely.
The film was then withdrawn, dipped in ultra-pure water which was
replaced with fresh water 4-5 times to remove all the unreacted HF.
Finally, the pellet was redispersed in water to obtain the individual
hollow PBzMA microspheres.

[0071]Fourier Transform Infra Red (FTIR) Spectroscopy

[0072]FTIR (Nicolet Magna-760, Nicolet Instrument Corporation, Madison,
Wis.) spectroscopy was used to identify a polymer on the bead surface and
also to ensure that silica was removed from the inside of the hollow
polymeric bead. Spectra were obtained at a resolution of 2 cm-1 and
averages of 64-100 spectra/scans (for enhanced signal) were obtained in
the wavenumber range 400˜4000 cm-1. Spectra of the pure silica
and polymer coated silica were recorded from KBr pellets, prepared by
mixing the microspheres with KBr in 1:100(wt/wt) ratio. FTIR spectra for
the pure PBzMA and the hollow polymer beads were obtained at room
temperature by casting a THF solution on KBr pellets. FTIR spectra of the
shell cross-linked hollow PBzMA microspheres were also measured from KBr
pellets, prepared by the same procedure described above.

[0073]Scanning Electron Microscopy (SEM)

[0074]SEM was performed using a JEOL SM 840 scanning electron microscope
(JEOL, Peabody, Mass.) at an accelerating voltage of 25 kV. Samples were
mounted on an aluminum stub and sputter coated with gold to minimize
charging. To obtain more information about the internal structure of the
hollow microspheres, dry etched polymer particles were sheared between
two glass slides after freezing in liquid nitrogen to obtain cracked
beads using standard procedures. This technique allows determination of
the polymer shell thickness.

[0079]Characterization of Hollow Polymeric Microspheres Produced by
Surface-Confined Living Radical Polymerization on Silica Templates

[0080]Spherical silica particles with an average diameter of 3μ were
used as a template for the synthesis of uniform hollow poly(benzyl
methacrylate) microspheres. The
((chloromethyl)-phenylethyl)trimethoxysilane (CTMS) initiator was
attached to the silica surface by treating the silica with CTMS in
acetone. Upon curing, a covalently linked benzyl chloride monolayer is
formed on the silica microsphere surface. Elemental analysis results
showed that the initial silica microparticles contained <0.02%
chlorine and that the CTMS-attached microparticles contained 3.15%
chlorine (Galbraith Laboratories, Inc., Knoxville, Tenn.). This
difference is equivalent to an average of 0.88 mmol initiator/g of
silica. The grafting density of the monolayer of benzyl chloride was 2.3
μmol/m2, calculated on the basis of average surface area (400
m2/g, data supplied by Phenomenex) of the pure silica particles. The
resulting surface modified silica particles could be redispersed in
organic solvents. Scanning electron micrographs of the CTMS modified
silica microparticles showed that they remain unaggregated (FIG. 1A) and
were similar to the original silica microparticles, exhibiting no
characteristic features. Although, benzyl chloride (--Ph--CH2Cl) of
CTMS is generally not an efficient initiating group for atom transfer
radical polymerization compared to 1-phenylethyl chloride or bromide, it
performed adequately in this case. Silica microspheres are coated with
higher molecular weight PBzMA by using alternative initiators such as
those listed in Table 3.

[0081]The surface modified microparticles were then used as
macroinitiators for benzyl methacrylate atom transfer radical
polymerization. Polymer growth was confined to the surface of
initiator-modified silica microspheres. The polymer coated silica
microspheres were dispersed easily in good solvents for poly(benzyl
methacrylate)(PBzMA). FTIR spectra of the resulting composite particles
showed bands corresponding to both poly(benzyl methacrylate) and silica.
A SEM micrograph of the polymer coated silica microspheres shows that the
polymer is uniformly coated over the silica surface (FIG. 1B). Tapping
mode atomic force microscopy was used to obtain more detailed information
about the surface topography. The AFM image of the surface of hollow
polymer microspheres shows that the surface was very smooth. The root
mean-square roughness (Rq) value is 8-10 nm. This value compares well
with Rq values for silanized non-porous silica microspheres. ATRP forms
primarily monodisperse polymer chains, with a uniform surface coating.
The thickness of the polymer layer increases with increasing
polymerization time at fixed monomer concentrations. Although the
possibility exists that when polymer chains are densely grafted to a
surface, steric crowding forces the chains to stretch away from the
surface, the curvature of the silica particles may help to reduce steric
crowding. Overall, the thickness of the polymer layer should be larger
than the radius of gyration for the equivalent free polymer in solution.

[0082]Polymer/silica particle composites were converted to hollow
polymeric microspheres by immersing a pellet of the composite particles
(supported by a Fluopore membrane) in an aqueous solution of HF. Silica
dissolution occurs via transport of etchant through the polymer shell to
the core. FIG. 2A shows the SEM micrograph of the aggregated intact
hollow PBzMA microspheres after etching the silica core. Aggregated
hollow polymer particles were redispersed as individual particles by
sonicating a portion of the pellet in water (FIG. 2B). When the composite
particles are prepared by 1 h polymerization, no hollow microspheres are
obtained after HF etching. This result indicates that the polymer shell
thickness was not sufficient to maintain the initial spherical structure
of the silica microsphere upon core removal. The hollow microspheres are
soluble in THF and other organic solvents because the polymer chains are
no longer grafted to the solid silica surface. This result proves that
the silica cores are completely etched by the HF solution. Shell
cross-linked hollow polymer microspheres, however, are not soluble in
most organic solvents. For this reason, they are useful for drug delivery
or encapsulating drugs/dyes in non-aqueous solvents.

[0083]After etching the silica core, the spheres were dissolved in THF and
the molecular weight of the dissolved polymer was determined by GPC. The
molecular weights of three samples of cleaved surface initiated PBzMA,
prepared with different polymerization times, are given in Table 4.

[0084]The molecular weight (Mn) of the grafted polymer, as determined by
GPC increased with polymerization time. The molecular weight distribution
(Mw/Mn) remained narrow after the initial stage of polymerization. The
polydispersity indices are consistent with that expected from living
polymerization (PDI<1.5) for the 6.5 h and 14 h cleaved samples,
although, the polydispersity of the 3.5 h sample is somewhat higher than
1.5.

[0085]In order to confirm that the microspheres were hollow, they were
frozen in liquid nitrogen and then crushed between two glass plates. FIG.
3 shows a SEM image of intact and broken polymer microspheres. Broken
hollow PBzMA microspheres produced by varying the polymerization time are
shown in FIGS. 4A-C. The shell thicknesses were measured from the SEM
micrograph of the broken hollow PBzMA particles, which are given in Table
4. The data reveal that shell thickness increases with increasing
polymerization time. Measured shell thicknesses of the samples prepared
with different polymerization times were higher than expected based on
the calculated values for the fully extended chains from their respective
molecular weights. Higher shell thickness values may be due to a number
possibilities. First, shell thickness was measured using SEM after the
hollow polymer microspheres were freeze-fractured. It is possible that
measured shell thickness is artificially high due to distortion of the
polymer since the microspheres were frozen and compressed between glass
plates prior to fracturing. A second possibility is the formation of
polymer inside the pores of the silica templates. Polymer chain
attachment and growth at different distances from the template center
contributes to the observed shell thickness after etching. The silica
core dissolution process may also affect the shell thicknesses. For
example, when HF diffuses through the polymer shell and reaches the core,
it reacts with silica to form silicon tetrafluoride gas and the polymer
chains detach from the surface at their point of attachment. The
resulting gas from the interior may produce micro voids inside the
polymer shell and result in an increased shell thickness. The detached
polymer chains have no solid support, and may also aid in the void
generation.

[0086]For further confirmation that the polymer microspheres contain
little or no silica inside the core, FTIR characterization was performed
on etched hollow PBzMA particles and was compared with pure poly(benzyl
methacrylate) and neat silica. FIG. 5 shows the FTIR spectra of pure
silica, PBzMA coated silica particles and PBzMA particles after etching
the silica core. Spectra for polymer-coated silica particles (plot b)
reveal bands at 750 and 697 cm-1 corresponding to phenyl C--H
out-of-plane bending, and benzene out-of-plane ring bending respectively,
and the 1728 cm-1 carbonyl stretching vibrations characteristic of
PBzMA. In addition to the PBzMA signals, a broad intense signal in the
1350-1000 cm-1 region corresponds to the solid state vibration of
the Si--O--Si bond in silica. The FTIR spectrum of the hollow PBzMA
particles (plot c) and cross-linked hollow particles (not shown) is
similar to the spectrum of neat PBzMA (plot d) and shows no spectral
characteristics of silica, confirming that the silica cores were etched
completely.

[0087]The data described herein demonstrate that the reliability and
predictability of a procedure for making uniform hollow microspheres
using surface-initiated controlled/living radical polymerization on
silica templates followed by core removal by etching. This method is
flexible and enables control over the shell thickness and composition by
adjusting polymerization time and monomer concentration. This approach is
useful for the fabrication of different shapes of hollow polymeric
materials produced from a variety of templates. The method is also useful
to produce hollow microspheres containing different polymer layers by the
sequential activation of the dormant chain in the presence of different
monomers during polymerization.

[0089]Colloidal assembly is a process by which particles ranging in size
from nanometers to micrometers are organized into structures by mixing
two or more particle types. Assembly is controlled by either specific or
non-specific interactions between particles. Examples include chemical
bonding, biological interactions, electrostatic interactions, capillary
action and physical adsorption. The assembly process is performed such
that smaller particles assemble around larger ones.

[0090]The colloidal assemby method described herein includes specific
chemical and biochemical interactions, which are manipulated to control
particle assembly. Polymer nanospheres are assembled onto the surface of
silica microspheres, and the assembled composite is subsequently heated
to a temperature above the Tg of the polymer nanospheres allowing the
polymer to flow over the silica microsphere surface, resulting in a
uniform core-shell composite. The methods used to assemble 100 and 200 nm
diameter amine-modified polystyrene (PS) nanospheres onto 3-10 μm
diameter glutaraldehyde-activated silica microspheres. SFM was used to
estimate the packing density of polymer nanospheres on the silica
microsphere surfaces. The biospecific interaction between avidin and
biotin was also used to control the assembly of PS nanospheres onto
silica microspheres. Avidin, a 40 kD glycoprotein, is known to have four
high affinity binding sites for the vitamin derivative biotin
(MW=244.31). When avidin-labeled PS nanospheres were mixed with
biotin-labeled silica in the appropriate number ratio, PS nanospheres
assembled onto the microsphere surfaces, covering the microspheres.
Thermally annealed composites, produced by heating PS nanosphere-silica
microsphere assemblies at temperatures higher than the Tg of PS, were
characterized using several analytical techniques. The compositions of
the resulting core-shell materials were confirmed by FTIR spectroscopy to
be PS-silica composites. The uniformity of the shell material coating was
confirmed by scanning electron microscopy (SEM). Core silica particles
were etched with hydrofluoric acid to confirm the existence of the shell
structure. The resulting hollow polymer microspheres were characterized
by transmission electron microscopy (TEM). Composite polymer shell
core-shell materials were also produced by mixing PS and poly
(methylmethacrylate) nanospheres in varying ratios prior to assembly and
annealing.

[0091]Uses for Core-Shell Composite Compositions

[0092]The shell material of a core-shell composite is used to allow
dispersal of the core composition in a particular solvent or to protect
the core from dissolution in the solvent. For example, core-shell
materials are prepared with polymer shells to protect medicines or other
materials from dissolution or hydrolysis. Polymer shells are used to
stabilize pigments in paints. Core-shell materials are also be useful to
strengthen polymeric materials. Other areas of application include the
preparation of stationary phases for chromatography or in the preparation
of sensing materials. For example, a thin polybutadiene film can be
physically adsorbed onto zirconia surfaces and then cross-linked,
resulting in a stationary phase for reversed-phase chromatography with
exceptional stability at high pH. Core-shell nanoparticles loaded with
gadolinium are useful as contrast agents for magnetic resonance imaging.

[0093]The following reagents and methods were used to construct
microspheres using colloidal assembly.

[0097]Amine-labeled silica microspheres were prepared for assembly by
first activating with glutaraldehyde. Prior to activation, approximately
2-6 mg of dry microspheres were placed into an eppendorf tube and washed
five times with 1.0 mL of ultra-pure water. Microspheres were cleaned
using five cycles of centrifugation, supernatant removal and resuspension
in 1.0 mL of ultra-pure water. Microspheres were centrifuged at
5,000×g. The microspheres were then washed two times with 1.0 mL of
50 mM phosphate buffer pH 6.9. The microspheres were then centrifuged
again (at 5,000×g), the supernatant removed, and 1.0 mL of a 2.5%
glutaraldehyde solution in 50 mM phosphate buffer pH 6.9 was added. The
microspheres were suspended, covered with foil and mixed on a vortex
shaker for two hours at 4° C. After two hours the microspheres
were washed five times with 1.0 mL of ultra-pure water. Microspheres were
washed another five times to exchange them into a 50 mM phosphate buffer
pH 7.4. The microspheres were stored at 4° C. protected from light
until needed for the assembly process. Amine-modified polystyrene
nanospheres were similarly prepared for assembly by washing (centrifuging
at 18,000×g and resuspending in water) five times with 1.0 mL of
ultra-pure water and two times with 1.0 mL of 50 mM phosphate buffer pH
7.4. Nanospheres were stored at 4° C. until they are used in the
assembly process. 100 mL of a 2.7% (w/v) suspension of nanospheres are
routinely prepared using this procedure.

[0098]Biotin-labeled silica microspheres were prepared by treating 2-4 mg
of amine-labeled silica microspheres with 1.0 mL of a 5 mM solution of
biotin-SE in 0.13 M sodium bicarbonate buffer pH 8.3. The microspheres
were suspended and shaken on a vortex shaker for one hour at 4° C.
Excess biotin-SE is removed with several cycles of centrifugation
(5,000×g), supernatant removal and resuspension with 1.0 mL of 50
mM phosphate buffer pH 7.4. The microspheres were stored at 4° C.
until used in the colloidal assembly process. Alternatively, silica
microspheres were labeled with avidin by treating 2-4 mg of
glutaraldehyde-activated silica microspheres with 1.0 mL of a 2 mg/mL
avidin in phosphate buffer pH 6.9 for 2 hours at 4° C. Excess
avidin was removed by several cycles of centrifugation and resuspension
in 50 mM phosphate buffer pH 7.4.

[0099]Biotin labeled nanospheres were prepared as follows: 100 mL of a
2.7% (w/v) suspension of amine-modified polystyrene nanospheres was
washed four times with 1.0 mL of ultra-pure water then with 1.0 mL of
0.13 M sodium bicarbonate buffer pH 8.3. Biotin-SE was then added to a
final concentration of 5 mM. The nanospheres were shaken on a vortex
mixer for one hour at 4° C. Subsequently excess biotin-SE was
removed with three cycles of centrifugation/resuspension in 1.0 mL of 50
mM phosphate buffer pH 7.4. When avidin-modified nanospheres were needed
for the assembly process, biotin-modified nanospheres (in 50 mM phosphate
buffer pH 7.4) were treated with 1.0 mL of a 0.1 mg/mL solution of avidin
in 50 mM phosphate buffer pH 7.4, The nanosphere/avidin suspension was
gently mixed and then shaken for two hours at 4° C. on a vortex
shaker. Subsequently, the nanospheres were washed seven times with 1.0 mL
of 50 mM phosphate buffer pH 7.4. Avidin-modified nanospheres were stored
at 4° C. until they were used in the assembly process.

[0100]Poly(Methyl Methacrylate)PMMA Nanosphere Preparation

[0101]Amine-modified PMMA nanospheres were prepared from carboxyl-modified
PMMA nanospheres by conversion of the carboxyl groups to a succinimidyl
ester and then treating the nanospheres with ethylenediamine. The
procedure was as follows: 100 mL of a 2.7% (w/v) suspension of PMMA
nanospheres (80 nm mean diameter) was washed five times with ultra-pure
water, then two times with 50 mM MES pH 4.75 containing 0.5% (w/v) NaCl.
Next 1.0 mL of a 10 mM NHS/60 mM EDC solution in MES buffer pH 4.75 was
added and the nanospheres were suspended and mixed on a vortex mixer on
low setting. Mixing continued for one hour at 4° C. in the dark.
After one hour, the nanospheres were centrifuged (18,000×g) and the
supernatant removed. One mL of fresh NHS/EDC solution was then added and
the nanospheres were suspended and mixed. Nanospheres were shaken at
4° C. for another hour. After this time, the nanospheres were
immediately centrifuged (18,000×g, 15 minutes), the supernatant was
removed and 1.0 mL of a 10 mM ethylenediamine solution in 50 mM phosphate
buffer pH 7.4 was added. The nanospheres were suspended and mixed on a
vortex shaker. The reaction was allowed to continue at 4° C. for
one hour. Following treatment with ethylenediamine, the nanospheres were
washed two times with ultra-pure water and then five times with 50 mM
phosphate buffer pH 7.4. Amine-modified PMMA nanospheres were stored at
4° C. until used in the assembly process

[0102]Nanosphere-Microsphere Assembly

[0103]The colloidal assembly process described herein was controlled by
either specific chemical or biochemical interactions. The reactions of
amine-modified polystyrene nanospheres with glutaraldehyde-activated
silica microspheres and avidin/biotin labeled polystyrene nanospheres
with avidin/biotin silica microspheres were used to direct the assembly.
Amine-modified PS nanospheres were assembled onto aldehyde-activated
silica microspheres as follows: Aldehyde-activated silica microspheres
(2-4 mg) were suspended in 50 mM phosphate buffer pH 7.4. Depending on
the particle sizes; this suspension contained microsphere concentration
of approximately 6.0×108 particles/mL. An appropriate volume
of a suspension of amine-modified PS nanospheres in phosphate buffer pH
7.4 was then added so that a 5000:1 number ratio of nanospheres to
microspheres was achieved. The suspension was shaken at 4° C. for
12-18 hours on a vortex mixer. Subsequently the product was purified by
alternately centrifuging (200×g) and resuspending the assembled
product in ultra-pure water. An identical process was followed when
assembly was controlled by the biospecific interaction of avidin and
biotin labeled nanospheres and microspheres.

[0104]Nanosphere-Microsphere Assembly Melting

[0105]In order to produce a material with a core-shell morphology the
nanosphere-microsphere assemblies were heated at 170-180° C. in
ethylene glycol using a temperature-controlled hot plate with a silicone
oil bath. As the temperature increased above the glass transition (Tg) of
the polymer nanospheres, the polymer melted and then flowed over the
surface of the silica microsphere templates. As a result, uniform
core-shell materials consisting of a silica core and polymer shell were
produced. In order to prepare the nanosphere-microsphere assemblies for
melting two milligrams of the assembled product was suspended in 250 mL
of ethylene glycol.

[0106]Ethylene glycol was chosen as the solvent because it has a high
boiling point and polystyrene is insoluble in it. This suspension was
then added to 750 mL of ethylene glycol in a glass vial maintained at
170-180° C. (silicone oil bath). The mixture was stirred
vigorously for 5-10 minutes. The mixture was then removed from the oil
bath and sonicated while cooling in room temperature water. The
suspension was centrifuged and resuspended in ethanol two times. The
suspension was dried on a piece of aluminum and subsequently was brought
to a temperature of 170-180° C. The composite was heated at this
temperature for 20 minutes. After this time the product was allowed to
cool, removed from the metal and resuspended in ultra-pure water. The
product was sonicated for two minutes, then centrifuged and resuspended
in ultra-pure water an additional two times. To melt the avidin-biotin
directed assembly, the composite was first washed with ethanol then
applied in a thin layer on an aluminum metal block. The block was then
heated at 170-180° C. for 20-30 minutes to allow the assembled PS
nanoparticles to melt and flow over the silica microsphere surfaces.

[0107]Electron Microscopy

[0108]SEM and TEM analysis was performed using standard techniques and
instrumentation.

[0109]Fourier Transform Infra Red (FTIR) Spectroscopy

[0110]FTIR (Nicolet Magna-760, Nicolet Instrument Corporation, Madison,
Wis.) spectroscopy was used to identify the polymer on the microsphere
surfaces. Spectra were obtained at a resolution of 2 cm-1 and
averages of 64-100 spectral/scans (for enhanced signal) were obtained in
the wavenumber range 400˜4000 cm-1. All samples were prepared
for analysis using a KBr pellet. Pellets were prepared using a 50:1
weight ratio of KBr to sample. All spectra were acquired at room
temperature.

[0116]The silica cores were etched using an 8% aqueous solution of
hydrofluoric acid. Approximately 1 mg of polystrene-silica composite was
suspended in 1.0 mL of ultra-pure water. Concentrated hydrofluoric acid
(50% w/v) was then added to bring the total HF concentration to 8%. The
suspension was allowed to stand for 20 minutes to assure complete removal
of the silica cores. The composite was then washed five times with 1.0 mL
of ultra-pure water. The resulting hollow polymer microspheres were then
air dried on glass slides prior to SEM or TEM analysis.

[0117]Characterization of Microspheres After Colloidal Assembly

[0118]The general procedure for the colloidal assembly of polymer
nanospheres with silica microspheres is shown in FIG. 12.

[0119]The assembly process was performed by mixing a suspension of
complementary types of nanospheres and microspheres at 4° C. for
12-18 hours. The assembly process was designed to pack as many
nanospheres onto the microsphere surface as possible. The numbers of
nanospheres to be packed on the microsphere surfaces was calculated by
dividing the theoretical microsphere surface area by the cross-sectional
area of a plane bisecting a nanosphere. The resulting value was used to
determine the minimum number of nanospheres needed in suspension for each
microsphere present. The number of nanospheres required to completely
cover a microsphere can be calculated using known methods, e.g., Ottewill
et al.,1997, Colloid Polym. Sci. 275: 274-283. The calculation is based
on hexagonal close packing of the nanospheres onto a planar surface.
Following assembly, the composites were heated at 170-180° C. to
melt the polymer nanospheres (FIG. 12).

[0121]The packing density of the polymer nanospheres on the surface of the
silica microspheres is an important variable in the formation of a
uniform polymer shell around the silica microspheres. Packing density was
easily and reliably controlled when the assembly process was controlled
by amine-aldehyde chemistry. The packing density was slightly more
variable when assembly was controlled by the interactions of avidin and
biotin-labeled colloidal particles. In some cases, aggregation may arise
during the synthesis of the avidin-labeled nanospheres, because the
nanospheres are first labeled with biotin and then subsequently treated
with an excess of avidin. Cross-linking of nanospheres may occur if the
concentration of nanospheres in the suspension is too high relative to
the amount of avidin used. Aggregation may also occur when insufficient
numbers of biotin-labeled nanospheres are mixed with avidin-labeled
silica microspheres. Despite these factors associated with the use of
avidin-biotin, the assembled composites are comparable in uniformity of
thickness to those formed when amine-glutaraldehyde is used to control
the assembly process.

[0122]Since the methods used to control the assembly process involve
specific chemical and biochemical interactions, it was necessary to
verify that the assembled composites were the result of these specific
interactions between the particles and not to non-specific interactions.
Non-specific binding during the assembly process was minimal for both the
amine-glutaraldehyde and avidin-biotin methods. Percentages of
non-specific binding were estimated based on the theoretical maximum
number of nanospheres that could cover one-half of a microsphere surface.
The number of nanospheres visible in SEM images of controls assembled
with non-specific binding were counted and taken as the percentage of the
theoretical maximum. Approximately 10-12% non-specific binding was
observed when amine-modified nanospheres were mixed with amine-coated
silica microspheres. The weight % nitrogen was 0.73% and <0.5% for the
amine-labeled silica microspheres and amine-modified polystyrene
nanospheres respectively. An SEM image of microspheres prepared using
non-specific binding (control) is shown in FIG. 15.

[0123]Other non-specific binding controls included mixing unmodified
polystyrene nanospheres with aldehyde-activated silica microspheres,
amine-modified polystyrene nanospheres with unmodified silica
microspheres and unmodified polystyrene nanospheres with unmodified
silica microspheres. In each of these cases, non-specific binding was
<1%. A non-specific binding control for avidin-biotin directed
assembly was performed by mixing avidin-labeled nanospheres with
avidin-labeled silica microspheres. Approximately 1% non-specific binding
was observed.

[0124]The assembled composites prepared by either the amine-glutaraldehyde
or avidin-biotin methods were very stable (as observed by SEM). No
noticeable changes in the surfaces of the materials were observed upon
several weeks storage in solution at room temperature or at 4-8°
C. Suspension in ethanol or ethylene glycol had no effect unless the
temperature was increased above the glass transition (Tg) of PS.
Stability of the assembled composites in ethylene glycol was important
since the melting procedure was performed at elevated temperature in this
solvent.

[0125]The polystyrene nanosphere/silica microsphere assemblies were heated
in ethylene glycol under the premise that the polymer nanospheres would
melt and the polymer would flow over the silica microsphere surfaces,
producing a core-shell composite with a uniform polymer coating. Ethylene
glycol was chosen as the solvent for heating the materials because it has
a high boiling point and because polystyrene and many other polymers are
insoluble in it. Microsphere aggregation during the heat treatment was
minimized by controlling the concentrations of microspheres in solution.
Annealing the composites on an aluminum metal block after the initial
heating in ethylene glycol helped to improve the uniformity of the
polymer coating. Melting of the 100 nm PS/3 μm silica microsphere
assembly (FIG. 3.3a) at high temperature in ethylene glycol, followed by
heating on an aluminum block results in the uniform PS-silica core-shell
composite shown in FIG. 13D. Avidin-biotin assembled composites could
only be melted on an aluminum metal surface because melting in an
ethylene glycol solution did not result in uniformly coated core-shell
composites. This result may be due to the instability of the
avidin-biotin linkage in solution at the high temperatures used to melt
the nanospheres, which causes them to dissociate.

[0126]To verify that polystyrene was coating the silica microspheres, two
independent evaluations were performed. A time study was conducted by
heating the polystyrene nanoparticle-coated silica microspheres in
ethylene glycol and then removing aliquots of assembled microspheres at
various times during the course of the 30-minute heating. SEM images
showed that nanoparticles remained attached to the silica microsphere
surface after five minutes. As the heating time increased, the spherical
nanoparticles melted and gradually filled in the spaces between
nanoparticles until the surface was uniformly coated. The integrity of
the polymer shells was determined by removing the silica cores by
chemical etching with hydrofluoric acid. After chemical etching, hollow
polymer shells were all that remained of the composite. The hollow
polymer microspheres produced remained intact after sonication in
ultra-pure water and centrifugation at 2,000×g. This result
provides additional evidence of polymer coating, since the silica
microspheres do not survive such treatment. A TEM of hollow polymer
shells produced by assembling 200 nm PS nanospheres onto 3 μm silica
microspheres, followed by annealing and chemical etching is shown in FIG.
16.

[0127]FTIR spectra (FIG. 17) of the PS-silica core-shell composites
provide additional evidence that the melting procedure results in polymer
coated microspheres. The methods described herein provide considerable
control in the assembly process to consistently yield core-shell
compositions and hollow micropheres in which the thickness of the shell
is essentially uniform, i.e., the thickness varies less than 10%.

[0128]The spectra reveal bands at 750 cm-1 and 697 cm-1, which
correspond to the phenyl C--H out-of-plane bending and benzene
out-of-plane ring bending respectively. Both of these resonances are
characteristic of polystyrene and are absent from the starting silica
microspheres. Aliphatic C--H stretching resonances of polystyrene (2900
cm-1) can be seen in the FTIR spectra shown in FIG. 18.

[0129]A 50:50 mix of PS and poly (methylmethacrylate) nanospheres resulted
in an assembled composite with a polymer nanosphere composition
corresponding to this ratio. This assembly was heated at 170-180°
C. in ethylene glycol to melt both nanosphere types assembled on the
silica microspheres. The resulting core-shell composite was confirmed by
FTIR spectroscopy to be a PS-PMMA composite (FIG. 19), The data described
herein indicate that assembled materials predictably produce core-shell
composites, e.g., those containing a silica core and a polystyrene shell
of essentially uniform thickness. The methods can be used to create a
shell that is a composite of multiple polymer types by mixing polymer
nanospheres in the ratio desired prior to assembly. Such materials have
applications in both analytical and materials chemistry development.
Core-shell composite materials are useful in the design of layered
sensing materials, the production of stationary phases for
chromatographic separations or the development of drug delivery systems.

[0130]Nanosphere/microsphere assembly accesses novel materials a reliable
and flexible procedure. By selecting the compositions of the particles
used in the assembly procedure, considerable control is gained over the
physical and chemical properties of the resulting composites. Additional
control over physical/chemical properties is achieved by the ability to
melt assembled polymer particles yielding uniform silica core/polymer
shell composite materials. The use of specific chemical/biochemical
interactions to control the assembly process of colloidal particles has
several advantages over the use of electrostatic interactions or
heterocoagulation to prepare core-shell composites. One advantage is that
a wider range of materials may be assembled when specific interactions
are used. For example, particles that are not charged or have the same
charge are assembled using this technique. Amine-modified PS nanospheres
are assembled onto amine-labeled silica by activating the silica surface
with a cross-linking dialdehyde. Another advantage is the improved
stability of the assembled products when covalent or strong biospecific
interactions are employed. The stability of the bonds between the
particles allows the use of a wider range of pH's, ionic strengths and
solvents in the assembly process.